Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Spatiotemporal expression of RNA-seq identified proteins at the electrode interface.

Implantation of electrodes in the brain can be used to record from or stimulate neural tissues to treat neurological disease and injury. However, the tissue response to implanted devices can limit their functional longevity. Recent RNA-seq datasets identify hundreds of genes associated with gliosis, neuronal function, myelination, and cellular metabolism that are spatiotemporally expressed in neural tissues following the insertion of microelectrodes. To validate mRNA as a predictor of protein expression, this study evaluates a sub-set of RNA-seq identified proteins (RSIP) at 24-hours, 1-week, and 6-weeks post-implantation using quantitative immunofluorescence methods. This study found that expression of RSIPs associated with glial activation (Glial fibrillary acidic protein (GFAP), Polypyrimidine tract binding protein-1 (Ptbp1)), neuronal structure (Neurofilament heavy chain (Nefh), Proteolipid protein-1 (Plp1), Myelin Basic Protein (MBP)), and iron metabolism (Transferrin (TF), Ferritin heavy chain-1 (Fth1)) reinforce transcriptional data. This study also provides additional context to the cellular distribution of RSIPs using a MATLAB-based approach to quantify immunofluorescence intensity within specific cell types. Ptbp1, TF, and Fth1 were found to be spatiotemporally distributed within neurons, astrocytes, microglia, and oligodendrocytes at the device interface relative to distal and contralateral tissues. The altered distribution of RSIPs relative to distal tissue is largely localized within 100µm of the device injury, which approaches the functional recording range of implanted electrodes. This study provides evidence that RNA-sequencing can be used to predict protein-level changes in cortical tissues and that RSIPs can be further investigated to identify new biomarkers of the tissue response that influence signal quality. STATEMENT OF SIGNIFICANCE: Microelectrode arrays implanted into the brain are useful tools that can be used to study neuroscience and to treat pathological conditions in a clinical setting. The tissue response to these devices, however, can severely limit their functional longevity. Transcriptomics has deepened the understandings of the tissue response by revealing numerous genes which are differentially expressed following device insertion. This manuscript provides validation for the use of transcriptomics to characterize the tissue response by evaluating a subset of known differentially expressed genes at the protein level around implanted electrodes over time. In additional to validating mRNA-to-protein relationships at the device interface, this study has identified emerging trends in the spatiotemporal distribution of proteins involved with glial activation, neuronal remodeling, and essential iron binding proteins around implanted silicon devices. This study additionally provides a new MATLAB based methodology to quantify protein distribution within discrete cell types at the device interface which may be used as biomarkers for further study or therapeutic intervention in the future.

A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II.

Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs.

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

SMN and symmetric arginine dimethylation of RNA polymerase II C-terminal domain control termination.

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. Here we show that a CTD arginine residue (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires protein arginine methyltransferase 5 (PRMT5) and recruits the Tudor domain of the survival of motor neuron (SMN, also known as GEMIN1) protein, which is mutated in spinal muscular atrophy. SMN interacts with senataxin, which is sometimes mutated in ataxia oculomotor apraxia type 2 and amyotrophic lateral sclerosis. Because POLR2A R1810me2s and SMN, like senataxin, are required for resolving RNA-DNA hybrids created by RNA polymerase II that form R-loops in transcription termination regions, we propose that R1810me2s, SMN, and senataxin are components of an R-loop resolution pathway. Defects in this pathway can influence transcription termination and may contribute to neurodegenerative disorders.

Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

RIPSeeker: a statistical package for identifying protein-associated transcripts from RIP-seq experiments.

RIP-seq has recently been developed to discover genome-wide RNA transcripts that interact with a protein or protein complex. RIP-seq is similar to both RNA-seq and ChIP-seq, but presents unique properties and challenges. Currently, no statistical tool is dedicated to RIP-seq analysis. We developed RIPSeeker (http://www.bioconductor.org/packages/2.12/bioc/html/RIPSeeker.html), a free open-source Bioconductor/R package for de novo RIP peak predictions based on HMM. To demonstrate the utility of the software package, we applied RIPSeeker and six other published programs to three independent RIP-seq datasets and two PAR-CLIP datasets corresponding to six distinct RNA-binding proteins. Based on receiver operating curves, RIPSeeker demonstrates superior sensitivity and specificity in discriminating high-confidence peaks that are consistently agreed on among a majority of the comparison methods, and dominated 9 of the 12 evaluations, averaging 80% area under the curve. The peaks from RIPSeeker are further confirmed based on their significant enrichment for biologically meaningful genomic elements, published sequence motifs and association with canonical transcripts known to interact with the proteins examined. While RIPSeeker is specifically tailored for RIP-seq data analysis, it also provides a suite of bioinformatics tools integrated within a self-contained software package comprehensively addressing issues ranging from post-alignments' processing to visualization and annotation.

The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline.

In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. Here, we show that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, we show that Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). Our results unveil a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila.

Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain.

Arginine methylation modulates diverse cellular processes and represents a molecular signature of germ-line-specific Piwi family proteins. A subset of Tudor domains recognize arginine methylation modifications, but the binding mechanism has been lacking. Here we establish that, like other germ-line Tudor proteins, the ancestral staphylococcal nuclease domain-containing 1 (SND1) polypeptide is expressed and associates with PIWIL1/Miwi in germ cells. We find that human SND1 binds PIWIL1 in an arginine methylation-dependent manner with a preference for symmetrically dimethylated arginine. The entire Tudor domain and a bifurcated SN domain are required for this binding activity, whereas the canonical Tudor domain alone is insufficient for methylarginine ligand binding. Crystal structures show that the intact SND1 extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1 in different orientations. This analysis explains how SND1 preferentially recognizes symmetrical dimethylarginine via an aromatic cage and conserved hydrogen bonds, and provides a general paradigm for the binding mechanisms of methylarginine-containing peptides by extended Tudor domains.

Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules.

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.